The dopamine transporter (DAT) clears extracellular dopamine thereby limiting its lifetime after release. The clearance process consists of DAT translocating dopamine from the outside to the inside of dopaminergic neurons. The DAT is a target for psychostimulant drugs which, as DA, are substrates taken up by DAT (amphetamine), or are blockers inhibiting substrate translocation (cocaine). Other biogenic amine transporters are the serotonin transporter (SERT) and the norepinephrine transporter (NET), targets for many different antidepressant drugs. Structural insight into these proteins has recently come from crystallization of a bacterial homolog, the leucine transporter LeuT, showing the location of the substrate binding site (termed S1 in the following), a region with high similarity among DAT, SERT, and NET. Novel evidence indicates the presence in LeuT of a secondary substrate site (S2) in the outer vestibule, playing a fundamental role in substrate uptake. In reversed uptake (efflux), one can postulate an S3 site in the inner vestibule, in symmetry to S2 in uptake. It is nt known whether S2 or S3 sites exist in DAT, SERT, or NET. We will study these sites in binding (dissociation) experiments, and in uptake and efflux assays. The impact will be assessed of disrupting S2 or S3 sites by mutating key residues. LeuT, which in preliminary experiments shows DAT-like properties, will be co-crystallized with bivalent ligands carrying two DA heads enabling them to bridge S1 and S2 sites. In addition, LeuT will be co-crystallized with bivalent leucine, and compounds will also be designed for bridging S1 and S3. Binding of bivalent ligands, if needed with additional modifications of LeuT, may promote an inward-facing state of leuT, the structural elucidation of which has been elusive so far. No LeuT crystals with substrate in S2 have yet been visualized to our knowledge. Although abundant evidence points to oligomeric assemblies of DAT and other biogenic amine transporters, their functional role is not clear. We will test the hypothesis that individual protomers in an oligomeric DAT assembly are not functioning independently, and establish positive or negative cooperative interactions. There is evidence that oligomerization and glycosylation of biogenic amine transporters are intimately linked, but there is contrasting evidence as to which comes first in maturation of the transporter as it moves from nucleus to endoplasmic reticulum to Golgi to surface. We will study the relationship between oligomerization, glycosylation, and trafficking of DAT. Tools will be Lec 4 cells that produce partially glycosylated transporter, a construct that lacks the three N-linked glycosylation sites, and an assortment of DAT mutants with coding variations in humans we have shown to be linked with Dopamine Transporter Deficiency Syndrome (DTDS), an inherited early-onset infantile parkinsonism-dystonia. The long-term objectives of the project are to better understand biogenic amine transporter structure-function, important for the action of compounds that target DAT, SERT, and NET: drugs of abuse, medications for attention-deficit hyperactivity disorder, environmental toxins, and antidepressants.